Scanning Specification Ordering

Wavelength Range 190-700nm CE4300 Dynamic Absorbance Detector

Optical Bandwidth 4nm – constant Wavelength range 190-700nm, 4nm constant optical
Digital Resolution 1nm bandpass, ultra fast wavelength scan (on-the-fly)
Wavelength Accuracy 1nm with auto peak triggering.
Wavelength Reproducibility ± 0.1nm Variable wavelength monitoring, dual wavelength
operation and programmable monitoring for up to
Absorbance Range 0.001 to 3A
6 wavelengths.
Scan Speeds Up to 1000nm/s – UV scan in 200ms Complete with instruction manual, power cable,
Baseline Flatness ± 0.002A over most of range signal cable and air test cell.
Second Order Filter Fast automatic insertion at 370nm Requires flowcell to be ordered.
Noise < ± 0.5 x 10–5 A, 230nm, 2 s T/C, using air test cell
Drift < ±0.3 x 10–4 A/h, 230nm, at equilibrium using air test cell CE4051
Overlayed Scans Up to 8 scans may be overlayed Column mounting panel with column cover.
Scan Trigger Start of peak, set Abs value, point of inflection or at peak
Number of Peak Scans Up to 100 peaks for a single elution CE4050
Scan Storage Up to 100 scans password protected Column mounting panel with column cover and
Rheodyne injection valve fitted with 20µL loop.
Multiple Scans of a Peak Up to 8 scans of any peak
Used with DataStream Data to PC, PC control, scan triggering, Lambda max. Flowcells
Software scans, peak purity and purity calculation, graphical
presentation, overlay, normalisation, disk storage, library 1200 07 70 8µL x 10mm pathlength
store and search, export, and many other functions. 1200 07 71 30µL x 10mm pathlength
1200 07 72 4µL x 5mm pathlength
1200 07 73 1mm pathlength preparative flowcell
Variable Wavelength Detection Specification 1200 07 74 12µL x 7mm pathlength
Other flowcells available on request
Wavelength Range 190-700nm
Wavelength Accuracy 1nm Printers
Wavelength Reproducibility ± 0.1nm
Absorbance Range 0.001 to 3A 8000 70 01 Dot matrix printer with signal cable
Absorbance Linearity Better than 1% 0.001 – 2A 8000 72 01 Colour inkjet printer with signal cable
Optical Bandwidth 4nm – constant 8000 73 01 Laser printer with signal cable
Dual Wavelength Operation Simultaneous measurement at 2 wavelengths and ratio CE4900
Wavelength Programming Measurements at up to 6 different wavelengths with a PowerStream software – provides PC workstation
time program and selectable sensitivity control of Cecil HPLC systems and data processing
Noise ± 0.5 x 10–5 A at 230nm, 2s T/C, using air test cell of chromatograms.
Drift ±0.3 x 10–4 A/h at 230nm at equilibrium, using test cell 9000 20 00
Noise – Dual Wavelength ± 3 x 10–5 A at 250/280nm DataStream Software - required for operation of the
Drift – Dual Wavelength ± 3 x 10–4 A/h at 250/280nm, constant temperature CE4300 in conjunction with a PC. No data
processing of chromatograms.
General Specification
Optical System Double Beam
Monochromator Littrow with 1200 l/mm holographic grating
Straylight < 0.02% at 220nm and 340nm
Detectors Two Silicon Diodes
Lamp Deuterium lamp with elapsed time indicator
Taper Beam Optics All energy collected to minimise RI effects
Self Test Automatic at switch-on; continuous monitoring of key
functions
Autozero By control panel or external trigger
Time Constant 0.1 second – 10 seconds, selectable System Requirements
Wavelength Calibration Automatic at switch-on using deuterium emission line.
Display Four-line display of parameters, menus, prompts, ● Computer with Pentium class processor
wavelength, absorbance etc. ● Pentium 200 MHz or better
Fast Number Entry By use of special number keys ● Microsoft Windows XP
Event Marking From keypad or by external trigger ● Microsoft Windows 2000
Method Storage For up to 30 methods, security protected ● 128 MB of RAM or more
Automatic Power-Up/Off Pre-programmed to save warm up time ● Hard-disk space required: 15 MB
Outputs Two absorbance outputs, or absorbance and absorbance ● CD-ROM drive
ratio, parallel printer, data available to PC. ● XGA or higher resolution monitor
Inputs Event, autozero ● Monitor resolution 1024 x 768 or better
Size and Weight 365 x 290 x 140mm, 17kg
● Microsoft Mouse or compatible pointer
Power Requirements 115V or 230V, 50/60Hz, 50VA

Cecil Instruments policy is one of continuous development. We therefore reserve the right to change specification without notice.

CECIL INSTRUMENTS LIMITED MILTON TECHNICAL CENTRE CAMBRIDGE CB4 6AZ ENGLAND
ISO 9001: 2000
TELEPHONE: 01223 420821 FAX: 01223 420475
CERTIFIED COMPANY
E-MAIL: info@cecilinstruments.com WEB SITE: www.cecilinstruments.com

12
 ULTRA FAST SCANNING
UV-VISIBLE DETECTOR

1

New Super Fast Performance
WaveQuest the CE4300 Dynamic Absorbance Detector
(DAD), is an ultra high speed scanning powerful
alternative to Photo Diode Array (PDA) detectors. HPLC
method development, purity determination, R & D and
Variable Wavelength Monitoring are all possible to the
highest quality. Dual wavelength monitoring and
wavelength programming are also provided.
The design is based on a double beam
spectrophotometer concept, with ultra fast wavelength
scanning over the range 190-700nm.
This dynamic scanning provides UV spectra in only
200ms, previously only possible using the static optical
system of a PDA detector.
Powerful signal processing software has been developed
which enables very high quality “on-the-fly” scans to be
triggered by eluting peaks without peak degradation.
Spectral data is transferred by Cecil’s DataStream
software to a PC workstation, where the same software
offers spectral manipulation and processing, library
facilities and the export of data to applications such as
Excel, Word etc. All data may be stored on disk.
Eluting peaks may be integrated at the same time as
purity and peak identification is carried out. Quantitative
evaluation and reporting is carried out by Cecil’s CE4900
PowerStream software, which is combined with
DataStream software.
One Detector Covers All UV Applications
A single beam Photodiode Array Detector (PDA), used for
method development and R & D, will often be replaced by
a dual beam UV variable wavelength detector (VWD), for
routine monitoring, when the developed method is
adopted, because of the poor drift performance of single
beam PDA detectors. Their performance can be as much
as 20 times worse. WaveQuest has a double beam optical
system with very high all round performance and the
lowest drift available.
The CE4300 is used as a high performance UV detector for
variable wavelength monitoring. WaveQuest, which is
less expensive than a PDA detector, therefore has a
double cost advantage.
2
Universal UV/Visible Detector
WaveQuest the ultra fast scanning Dynamic Absorbance
Detector (DAD) is a major advance in UV-Visible detector
design, offering the capabilities of a Photo Diode Array
(PDA) detector with greatly improved performance when
used for chromatogram monitoring.
Very high speed scanning of eluting peaks, “on-the-fly”,
makes purity checks, the determination of optimum
monitoring wavelengths for unfamiliar peaks (lambda
max scans), and identification of peaks using library
spectra all possible, with considerable cost saving.
CE4300 Dynamic Absorbance Detector
WaveQuest the new CE4300 Dynamic Absorbance
Detector for ultra fast scanning, part of the Adept HPLC
range from Cecil Instruments, offers all the spectral
facilities of Photo Diode Array detectors, with better
noise and drift performance, particularly important when
used to monitor and quantify chromatograms.
The CE4300 has a double beam optical system in which
the dispersed radiation is dynamically scanned across
the detectors, in the manner of a UV-Visible double beam
spectrophotometer, at ultra fast speeds – up to
1000nm/second.
All components of the detector – monochromator, optical
system, lamp optics, detector optics and signal
processing software have been carefully optimised to
provide unprecedented performance in both wavelength
scanning and single or multi-wavelength monitoring.
The lower noise level when monitoring a chromatogram
makes accurate measurements at the highest
sensitivities possible when measuring very low
concentrations.
The lower drift, as much as 20 times lower, gives flat
chromatogram baselines making integration of peaks
more accurate, particularly at high sensitivities.
Taper Beam Optics
Taper beam optical imaging at the flowcell, with all
transmitted energy reaching the detector, provides
maximum energy collection. The result is minimal
baseline wander due to changes in the solvent refractive
index during gradient formation.
Performance Validation
Validation of detector performance is carried out
conveniently, using the instruments standard software.
A Holmium Oxide filter is automatically inserted in the
optical path when the calibration routine is selected.
Bandwidth validation is automatic, using the Deuterium
emission line at 656.1nm. Absorbance accuracy is
checked using filters traceable to NPL standards.
PowerStream Software – CE4900
PowerStream is a chromatography control and data
processing software and interface developed by Cecil
Instruments to be comprehensive and convenient to use
from a single PC workstation. It provides powerful data
handling and system control for isocratic, binary and
ternary HPLC systems.
DataStream software provides sophisticated spectral
processing. Spectral data is transferred from the detector
to the PC where it is graphically displayed, manipulated
and exported as required.
Chromatographic peaks are automatically scanned to
determine purity, identify components, and determine
optimum wavelengths for chromatogram monitoring –
Lambda max scans. Multiple scans of peaks enable
checks to be made of peak homogeneity.
Wavelength scans are compared with your own library to
establish the identity of unknown components. A spectral
library search facility is provided.
Visual comparison of spectra is valuable for identifying
components and for comparing scans made for purity
determination. To further assist assessment, a
mathematical calculation is provided, which produces a
figure indicating the degree of match or similarity.
New Advanced Design
The design of the WaveQuest, CE4300, Dynamic
Absorbance Detector is based on a double beam UV/Visible
Wavelength Scanning Spectrophotometer concept, with an
optical system which also provides the highest
performance as a dual wavelength, programmable detector.
The 4nm optical bandwidth, constant over the 190-700nm
range, provides high quality spectral scans for easy
identification of unknown components. The digital signal
processing and optical and mechanical systems provide
ultra fast scanning and the best noise and drift
performance available. Accurate measurements at high
sensitivities are therefore possible.
Ultra Fast Scans of Narrow Peaks
The ultra high speed scanning of the WaveQuest is
shown. A very fast binary separation was carried out, on
a Cecil gradient elution system 4S, of sixteen
polyaromatic hydrocarbons, designated by the American
Environmental Protection Agency as priority pollutants.
The time for the elution was less than 4 minutes, using a
5cm column with a 3 micron packing.
Several of the peaks have widths at half height less than
1.5 seconds. The spectra of all peaks were displayed in
real time and are shown here overlayed in groups of four.
3
Peak Impurities Revealed
The outstanding performance of the WaveQuest is
illustrated by a number of chromatographic
applications described in this brochure.
The application shown here is a chromatogram of a
mixture, obtained under isocratic conditions. It
appears to be a simple three component mixture.
Peak purity checks were made for each of the peaks
with ultra-fast “on-the-fly” scans on both the
ascending and descending sides of each peak.
Peaks 1 and 2 gave identical spectral scans but Peak 3
gave totally different spectra between the “up side”
and “down side” scans indicating a major peak
impurity, giving rise to the scan shown in red.
Despite the presence of the impurity, peak 3 appears
visually symmetric and indeed the asymmetry factor
calculated for the peak was close to 1.00, giving no
indication of the presence of an impurity.
4
Dual High Performance
When a method has been developed using the CE4300
Dynamic Absorbance Detector, the detector may be
used as a sensitive and very stable monitor for the
application, with performance much superior to that of
a Photo Diode Array detector.
Should the CE4300 be required for further method
development or R & D it may be replaced by the
CE4200 Variable Wavelength Monitor. This is a top
performance detector with superb noise and drift and
is therefore recommended for detection at the very
highest sensitivities. Excellent linearity allows
accurate quantification of samples at very low or high,
concentrations.
WaveQuest offers better sensitivity and lower
detection limits than a Photo Diode Array detector.
The WaveQuest represents two instruments in one, a
scanning spectrophotometer and a variable
wavelength detector, resulting in major cost saving.
Lambda Max Scans
The same trial mixture as before, which separated into
three peaks under isocratic conditions, separated into four
peaks under binary gradient conditions using an Adept
System 4S.

Each peak was scanned at its maximum - Lambda max scan

– and the spectra displayed whilst the chromatogram was
simultaneously displayed and quantified by integration to
provide baseline corrected peak areas and peak heights.

Peaks 1, 2 and 4 are quantitatively similar and all have peak

maxima close to 222 and 273nm.

Peak 3 scan is clearly very different having maxima near

234, 268, 316, and 331nm.

Optimum monitoring wavelengths for each peak may be

chosen from these scans.

The photo shows an Adept Isocratic System 2S. The

WaveQuest detector, fitted with a column and injector valve
panel, is mounted on the CE4100 pump, with the
PowerStream interface unit at the bottom of the stack.

An optional column oven is fitted on the right. The

system offers Diode Array performance and the very best
dual and variable wavelength monitoring, with
exceptional compactness, as does the Adept Gradient
System 4.
5
Normalised Overlays Confirm Purity
Automatic peak purity checks were made by the same
upside and downside, “on-the-fly”, scans for all four
peaks.

The results are shown for peaks 3 and 4 only, overlayed

before and after normalisation of the spectral scans. Peak
4 is shown in the upper two plots and Peak 3 in the lower
two.

From the overlays, particularly when normalised, it is

seen that a near perfect match is obtained indicating that
these peaks were homogenous and hence pure, as also
were Peaks 1 and 2.

Eight Scans of Peaks

Several means of triggering the wavelength scan of a
peak are user selectable.

In the example shown here, WaveQuest was

programmed to make repetitive scans of pyrene as it
eluted.

The use of the cursor is demonstrated by using it to

measure the absorbance maximum at 326.7nm. The drop
line shows that all the scans have the same wavelength
for this absorption maxim.
6
 Spectral Overlay Confirms Identity
A different mixture of the same components was used to
produce the chromatogram shown here with its 4 peaks.

Spectral library searches were made in order to identify

the components, in conjunction with retention time data.

Peaks 1, 2 and 4 can be identified as alkyl phthalates, most

probably dimethyl, dibutyl and di(ethylhexyl) phthalates.

ADVANCED DESIGN Peak 3 is identified as pyrene. The library reference

spectrum of pyrene overlayed on this impurity peak
● 190-700nm shows an almost perfect fit, and together with the
● Ultra fast “on-the-fly” scans retention time data for pyrene provides confirmation of
identity.
● UV scan in 200 ms
● Double beam
● 4nm optical bandpass
● 1nm digital resolution
● Superb stability
● Fast warmup

7
Analysis of Sugar Free Soft Drinks
The new ultra fast scanning detector from Cecil
Instruments, the WaveQuest, was used as part of an
Adept System 4S to separate, identify and quantify the
components of soft drinks.

Using the CE4300, high quality UV-Visible spectra were

obtained “on-the-fly” without the use of an expensive
Photo Diode Array detector. VERSATILITY
The spectra indicate the most appropriate wavelength for ● Highest sensitivity VWD
monitoring any given material. The spectra together with
retention time data are sufficient to provide positive ● Very low noise
identification of eluted peaks.
● Very low drift
The chromatographic separation of commonly occurring
● Method storage
components of soft drinks and fruit juices is shown. The
gradient elution used a C18 column, with a phosphate ● Dual wavelength operation
buffer: methanol binary gradient, monitored at 210nm as
some of the components lack chromophores at longer ● Programmed monitoring of 6 wavelengths
wavelengths.
● Simultaneous 4 wavelength monitoring
The peaks in the chromatogram were scanned “on-the-
fly” during elution and the spectra displayed in adjacent
windows.

Four scans are shown here, overlayed. Sufficient

information is available to identify a component peak
when taken in conjunction with the retention time.
8
Library Search Yields Identification BENEFITS
The use of the Spectral Library for peak identification is
● Spectral Storage
demonstrated by the chromatogram shown, which was ● Simple intuitive menu operation
obtained from a low cost “lemonade” style soft drink.
● Stackable with Adept HPLC modules
Comparisons were made between the UV spectra of the ● Flowcell replacement without adjustment
eluted peaks, with those of reference spectra which had
been previously scanned and stored in the Spectral ● Validation software
Library. ● Spectral library
Overlay of these scans after normalisation indicated that ● Library search
these peaks were identical with Citric Acid (Peak 3),
Saccharin (Peak 5), Aspartame (Peak 7) and Benzoic Acid
(Peak 9).

Retention times for the major components coincided with

those of the library spectra confirming the identifications.

The scan of Peak 9 is shown overlayed with the Library

Spectrum of Benzoic Acid displayed in the upper window.
The very high quality of the spectral match obtained, is
clearly seen.

A statistical calculation is programmed in the DataStream

software, enabling a match index to be calculated
between spectra, for identification and also purity.

9
Spectral Match Index
To confirm the identity of the sixteen separated peaks
shown in the fast separation, known reference standards
were injected and eluted under the same conditions as
the sample mixture.

Comparison of spectra and retention times allows

positive identification of all the components.

The screen display shown here illustrates the process,

showing a reference library spectrum, which is overlayed
on peak 8 in the chromatogram of the sixteen component
mixture.

Applying the mathematical routine for spectral match, or

similarity, to the data for the sample and library spectra
shown here, produced a match index of 996.5, which is
displayed on the screen.
A figure greater than 990 indicates a good degree of
match, and a figure above about 900 indicates some
degree of match.
A figure less than 100 indicates very little match, or
similarity between the scans.
The photograph shows the WaveQuest detector
fitted with the column mounting panel and cover. A
Rheodyne injection valve is also fitted to the panel
The arrangement provides close coupling between
the column and flow cell, for minimum dead
volume.

10
Excellent Analytical
Precision
The excellent precision of the
WaveQuest, and indeed the
complete Adept Gradient System
4S, is demonstrated by 7 repeat
Component Summary
injections and elutions of the soft
drink shown here in overlay mode,
each chromatogram being offset
by 50mA.

The phosphate buffer: methanol

baseline was first recorded and is
shown.

Statistical analysis of some of

these results is shown in summary
report comment.

The report shows a % RSD for the

peak caffeine of 0.33.

11